Ex vivo 3D osteocyte network construction with primary murine bone cells

Abstract

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Ex vivo construction of human primary 3D–networked osteocytes

“…In comparison to our previous results on murine long bone samples,[32] it took a less number of digestion cycles to isolate cells from human bone fragments. Also, the number of isolated cells became significantly less after the eighth digestion cycle (i.e., “D8”) for the human samples.…”

“…1, we previously reported[32] that the physiological morphology and biological functions of murine primary osteocytes can be replicated ex vivo by their 3D network construction in microfluidic culture chambers. We used a microbeads-guided assembly approach that: (1) consists of 3D cellular network of primary osteocytes, and (2) mitigate the rapid osteoblastic dedifferentiation and proliferation of primary osteoblast-like encountered in conventional 2D culture.…”

“…We used a microbeads-guided assembly approach that: (1) consists of 3D cellular network of primary osteocytes, and (2) mitigate the rapid osteoblastic dedifferentiation and proliferation of primary osteoblast-like encountered in conventional 2D culture. [32], [33] In this biomimetic assembly approach[32], [34]and as illustrated in Fig. 1b, biphasic calcium phosphate (BCP) microbeads of 20–25 µm were used to: (1) spatially distribute osteocyte cell bodies into the interstitial spaces between the microbeads with one cell occupying each interstitial site while allowing them to develop processes with neighboring cells with the physiologically relevant lacuna and interlacunar dimensions and (2) provide a mechanically stable framework to maintain the microscale geometry and dimensions of the 3D cellular network during perfusion culture.…”

Well plate-based perfusion culture device for tissue and tumor microenvironment replication

“…Therefore, our results suggested that the osteocyte phenotype of the harvested cells could be preferentially preserved inside of the tissue structure due the 3D confinement effect of microbeads as mentioned earlier. As it is beyond the scope of this device-centric paper, we report elsewhere 30 in details that these reconstructed osteocytes express SOST gene which produces sclerostin, as determined by both PCR and histological immunostaining. In contrast, this key osteocytic gene expression is lost within a few weeks of conventional 2D culture, as the osteocytes differentiate to osteoblasts.…”

An In Vitro Model for the Development of Mature Bone Containing an Osteocyte Network

“…Although primary osteoblast to osteocyte cell transition has recently been reported in 3D by a few research groups, our model differs in the close recapitulation of complex in vivo conditions and in the self-structuring process as opposed to a preformed organic-inorganic matrix template (e.g., collagen scaffold with embedded HA particles). [45,54,55] As such, our constructs survive over much greater periods of time (i.e., 12 months compared to a few weeks); as well as developing over a real tissue length scale. We have carried out a pilot study over 3 weeks in culture to test novel compounds that can inhibit the ossification process, obtaining promising results.…”